Synthesis, biological evaluation and molecular modeling of novel thienopyrimidinone and triazolothienopyrimidinone derivatives as dual anti-inflammatory antimicrobial agents

Article abstract of DOI:10.1016/j.bioorg.2017.12.028

New thienopyrimidinone and triazolothienopyrimidinone derivatives have been synthesized. These compounds were subjected to anti-inflammatory and antimicrobial activity screening aiming to identify new candidates that have dual anti-inflammatory and antimicrobial activities. Compounds 5, 7 and 10a showed minimal ulcerogenic effect and high selectivity towards human recombinant COX-2 over COX-1 enzyme. Their docking outcome correlated with their biological activity and assured the high selectivity binding towards COX-2. In addition, they could act safely up to 80 mg/kg orally or 40 mg/kg parentrally. The antimicrobial screening showed that compound 10a displayed distinctive inhibitory effect on the growth of Escherichia coli comparable to that of ampicillin. Moreover, compounds 5, 7, 9 and 12a possessed 50% of the inhibitory activity of ampicillin against E. coli. Thus, compounds 5, 7 and 10a represent promising dual acting anti-inflammatory and antimicrobial agents. This work provides rewarding template enriching the chemical space for dual anti-inflammatory anti-microbial activities.

Full text of DOI:10.1016/j.bioorg.2017.12.028

Accepted Manuscript
Synthesis, Biological Evaluation and Molecular Modeling of Novel Thienopyr-
imidinone and Triazolothienopyrimidinone Derivatives as Dual Anti-Inflam-
matory Antimicrobial Agents
Adnan A. Bekhit, Ahmed M. Farghaly, Ragab M. Shafik, Mona M.A. Elsemary,
Alaa El-Din A. Bekhit, Aida A. Guemei, Mai S. El-Shoukrofy, Tamer M.
Ibrahim
PII:
S0045-2068(17)30921-5
DOI:
https://doi.org/10.1016/j.bioorg.2017.12.028
YBIOO 2209
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 December 2017
28 December 2017
29 December 2017
Please cite this article as: A.A. Bekhit, A.M. Farghaly, R.M. Shafik, M.M.A. Elsemary, A.E-D. Bekhit, A.A. Guemei,
M.S. El-Shoukrofy, T.M. Ibrahim, Synthesis, Biological Evaluation and Molecular Modeling of Novel
Thienopyrimidinone and Triazolothienopyrimidinone Derivatives as Dual Anti-Inflammatory Antimicrobial
Agents, Bioorganic Chemistry (2017), doi: https://doi.org/10.1016/j.bioorg.2017.12.028
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Synthesis, Biological Evaluation and Molecular Modeling of Novel
Thienopyrimidinone and Triazolothienopyrimidinone Derivatives as Dual Anti-
Inflammatory Antimicrobial Agents*
1
1
1
1
Adnan A. Bekhit , Ahmed M. Farghaly , Ragab M. Shafik , Mona M. A. Elsemary , Alaa El-Din A.
2
3
1
4
Bekhit , Aida A. Guemei , Mai S. El-Shoukrofy and Tamer M. Ibrahim
1
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Alexandria University, Alexandria,
2
1521, Egypt.
2
Department of Food Sciences, the University of Otago, Dunedin, New Zealand
Department of Clinical Pharmacology, Faculty of Medicine, Alexandria University, Alexandria,
3
Egypt
4
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-
Sheikh 33516, Egypt
Running title: Novel Thienopyrimidinone and Triazolothienopyrimidinone Derivatives
Keywords: Thienopyrimidinone and triazolothienopyrimidinone derivatives, anti-inflammatory,
antimicrobial and molecular docking
*
This work is dedicated to the memory of Professor Ragab M. Shafik
Correspondence: Adnan A. Bekhit, Professor
E-mail address: adnbekhit@pharmacy.alexu.edu.eg
Tel.: +20-3-4871317; Fax: +20-3-4873273
1

Abstract
New thienopyrimidinone and triazolothienopyrimidinone derivatives have been synthesized. These
compounds were subjected to anti-inflammatory and antimicrobial activity screening aiming to
identify new candidates that have dual anti-inflammatory and antimicrobial activities.
Compounds 5, 7 and 10a showed minimal ulcerogenic effect and high selectivity towards human
recombinant COX-2 over COX-1 enzyme. Their docking outcome correlated with their biological
activity and assured the high selectivity binding towards COX-2. In addition, they could act safely
up to 80 mg / kg orally or 40 mg / kg parentrally. The antimicrobial screening showed that
compound 10a displayed distinctive inhibitory effect on the growth of Escherichia coli comparable
to that of ampicillin. Moreover, compounds 5, 7, 9 and 12a possessed 50% of the inhibitory activity
of ampicillin against E. coli. Thus, compounds 5, 7 and 10a represent promising dual acting anti-
inflammatory and antimicrobial agents. This work provides rewarding template enriching the
chemical space for dual anti-inflammatory anti-microbial activities.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) keep on to be the main treatment of inflammatory
diseases. However, they have been linked with many adverse effects such as gastrointestinal
mucosal damage and bleeding [1]. The discovery of the inducible enzyme COX-2 has pushed the
searching for anti-inflammatory agents forward that specifically target COX-2 and avoid the
undesirable side effects accompanied by treatment with classical non selective NSAIDs [2].
Marketed COX-2 inhibitors have an exceptional importance as a potent and gastrointestinal safe
anti-inflammatory agent [2]. Furthermore, the increasing microbial resistance to currently available
antimicrobial agents has reached an alarming level in many countries [3]. There has been a rapid
spread in the primary and opportunistic fungal infections because of the increased number of
immunocompromised patients. Candida albicans is one of the most common opportunistic fungi
responsible for such infection [4].
The co-administration of multiple drugs for treating inflammatory conditions associated with
microbial infections might amend some secondary health problems particularly in patients with
impaired kidney and / or liver functions. Therefore, from pharmacoeconomic perspective, and to
better patient compliance, a dual anti-inflammatory / antimicrobial agent with minimal adverse
effects and high safety margin is greatly needed. Significant confirmation accumulated in literature
showed the efficacy of thienopyrimidine derivatives as anti-inflammatory [5-9], and antimicrobial
agents [10-14].
2

Inspired by the above mentioned findings and as an extension of our ongoing program to synthesize
compounds with dual antimicrobial / anti-inflammatory activities [15-21], new thienopyrimidinone
and triazolothienopyrimidinone derivatives were prepared. This would enrich the chemical space
for the desired activities,[22] and provide rewarding template for further lead optimization efforts in
the future. The synthesized compounds were screened for their possible anti-inflammatory and
antimicrobial activities aiming to identify dual acting candidates.
Results and Discussion
Chemistry
Synthesis of the intermediate and target compounds was accomplished according to the
steps depicted in Scheme 1. The starting key compound was 3-Benzyl-2-sulfanyl-5,6,7,8-
tetrahydrobenzo[b]thieno[2,3-d]pyrimidin-4(3H)-one 2.
Scheme 1. Synthesis of some thieno- and triazolothienopyrimidinone derivatives 2-12. Reagents
and conditions: (i) C
6
H
5
CH
2
NCS, DMF, NaOH; (ii) CH
3
I ; (iii) NH
2
NH
2
.H O 98% ; (iv)
2
3

ClCOOC
CO ; (viii) = C
BrCH COOC /C
2
H
5
; (v) HCOOH; (vi) CS
NHCSNHNH
ONa; (xi) = 4-RC
2
, NaOH or RNCS, R = C
; (ix) = RX; (x) = ClCH
COCH Br.
6
H
5
, 4-CH
3
C
6
H
5
4
; (vii) ClCH
/ DMF/ K
2
CN, KI,
CO or
K
2
3
6
H
5
2
2
COOC
2
H
2
3
2
2
H
5
2
H
5
6
H
4
2
Synthesis of 2-sulfanyl derivative 2 was reported [23, 24]. However, it was prepared in our
laboratory starting from the aminoester 1 using a new method that was adopted for the preparation
of other similar derivatives [25]. In addition, methylsulfanyl derivative 3 was also prepared in our
laboratory via S-alkylation of compound 2 using methyl iodide[21].
Moreover, hydrazinolysis of compound 3 to give 2-hydrazino derivative 4 was adopted by refluxing
2
-methylsulfanyl derivative 3 with hydrazine hydrate 98 % [21]. Reaction of the 2-hydrazino
derivative 4 with ethyl chloroformate in dry pyridine yielded the open chain counterpart 5. Trials to
cyclize the open chain counterpart 5 to the corresponding oxotriazolo derivative either by increasing
1
the reflux time in pyridine or by heating above its melting point were unsuccessful. H-NMR
spectrum of compound 5 revealed two deuterium exchangeable singlets each integrated for one
proton due to two NH groups. The spectrum also exhibited a triplet and a quartet assigned for
CH
2
CH protons.
3
Moreover, treating the hydrazino derivative 4 with formic acid or carbon disulfide / sodium
hydroxide yield the cyclized unsubstituted triazolo derivative 6 [21] or the 1-sulfanyltriazolo
derivative 7, respectively. In addition, reaction of the 2-hydrazino derivative 4 with isothiocyanate
derivatives did not yield the corresponding phenylthiosemicarbazone derivatives. Instead,
1
compound 7 was obtained. This is in agreement with previously reported data [26-28]. H-NMR
spectrum of compound 7 showed a downfield deuterium exchangeable singlet characteristic for the
thiol group. Electron impact mass spectrum (EI-MS) of compound 7 showed the molecular ion peak
+
.
(
M ) at m/z 368 and the base peak at m/z 91 most probably corresponding to the tropylium
carbocation.
The sulfanyl derivative 7 was alkylated using chloroacetonitrile, alkyl or aralkyl halides, ethyl
chloroacetate or phenacyl bromides to the corresponding sulfanyl acetonitrile derivative 8, S-alkyl
or S-aralkyl derivatives 10a-c, ethoxycarbonylmethylsulfanyl derivative 11 or aroylmethylsulfanyl
derivatives 12a and 12b. Compound 8 was converted into phenylaminothiadiazolylmethylsulfanyl
1
derivative 9 by reaction with phenyl thiosemicarbazide. H-NMR spectrum of compound 8 lacked
the singlet due to SH proton and showed a new singlet integrated for two protons, assigned for
+
.
acetonitrile protons. EI-MS of compound 8 showed the molecular ion peak (M ) at m/z 407 and the
1
base peak at m/z 91 most probably corresponding to the tropylium carbocation. H-NMR spectrum
of compound 9 showed a multiplet at the aromatic region integrated for nine protons, assigned for
phenyl and three benzyl protons in addition to the deuterium exchangeable NH proton. A doublet of
1
two protons assigned for other benzyl protons was also detected. Moreover, H-NMR spectrum of
4

compound 10a lacked the singlet due to thiol proton and showed a new upfield singlet integrated for
three protons, assigned for the methylsulfanyl protons. EI-MS of compound 10b showed the
+
.
molecular ion peak (M ) at m/z 396 and the base peak at m/z 91 most probably corresponding to
+
.
the tropylium carbocation. EI-MS of compound 11 showed the molecular ion peak (M ) at m/z 454
1
and the base peak at m/z 91 most probably corresponding to the tropylium carbocation. H-NMR
spectrum of compound 12b lacked the singlet due to thiol proton, while showed a singlet integrated
for two protons assigned for the methylenesulfanyl protons and two doublets each integrated for
1
two protons due to the four benzoyl protons. Representative H NMR, mass and IR spectral data of
some compounds are included in the Supporting Information (SI).
Biological screenings
Anti-inflammatory Screening
Cotton pellet-induced granuloma bioassay[29]
The anti-inflammatory activity of the newly synthesized compounds was evaluated using the
cotton-pellet granuloma bioassay in rats and indomethacin was used as a reference compound. The
ED50 values were determined for each compound (Table 1).
Out of test compounds, compounds 7 and 10a exhibited anti-inflammatory activity that was more
potent than that of indomethacin. Compound 5 was nearly equipotent to indomethacin at the same
dose level and test conditions. Furthermore, compounds 6 [21], 10b, 10c, 11 and 12a exhibited
moderate anti-inflammatory activity. The rest of the compounds were found to be weakly active or
inactive as anti-inflammatory agents.
Ulcerogenic effects [30]
All compounds were evaluated for their ulcerogenic potential in rats. Indomethacin was used as
reference standard. All test compounds proved to have a superior gastrointestinal safety profile (0 -
3
0 %) in the population of the test animals at oral doses of 30 µmol / kg / day, compared with
indomethacin which was found to cause 100% ulceration under the same experimental conditions
Table 1). Gross observation of the isolated rat stomachs showed a normal stomach texture for
(
compounds 5, 7, 10a, and 10c (0 % ulceration). On the other hand, slight hyperemia (10 %
ulceration) was observed with compounds 9, 10b and 12b.
Human COX-1 and COX-2 enzymatic assay
Compounds 5, 7 and 10a that exhibited potent anti-inflammatory activity were additionally tested
for their ability to inhibit human COX-1 and COX-2 enzymes in-vitro by applying the methodology
of Wakitani et al.[31] and Hammarström and Falardeau.[32] The concentration of the compound
causing 50% enzyme inhibition (IC50 μM) was determined. Results are shown in Table 1.
5

Results indicated that test compounds were less active than indomethacin against COX-1. However,
they showed better inhibitory activities than indomethacin against COX-2, indicating that they are
selective inhibitors for COX-2. The approximate selectivity ratios (COX-1 / COX-2) of these
compounds were compared to that of the standard COX-2 selective inhibitor, Celecoxib. The result
showed that compound 10a was the most selective with approximate selectivity ratio of 192.3, yet it
had lower selectivity compared to that of Celecoxib (approximate selectivity ratio of 333). More
details on rationalizing the activity of compounds 5, 7 and 10a are discussed in the Molecular
Modeling section.
a
a
Table 1. The anti-inflammatory activity (ED50, μmol) , ulcerogenic activity (%) and the in-vitro
b
c
human COX-2 and COX-1 enzymes inhibitory activities of the test compounds.
COX-2
IC50
COX-1
IC50
Approximate
Selectivity Ratio
COX-1 / COX-2
ED50
μmol)
Ulcerogenic
activity %
Comp. No.
Celecoxib
(
d
d
(
μmol)
(μmol)
---
---
100
0
20
0
30
10
0
10
0
< 0.3
2.63
1.42
---
1.86
---
---
0.52
---
---
---
---
---
> 100
0.26
94.66
---
88.94
---
---
> 100
---
---
---
---
---
333
0.098
66.66
---
47.81
---
---
192.3
---
---
---
---
---
Indomethacin 9.76±0.12
5
9.36±0.24
19.54±0.18
9.16±0.16
16.36±0.22
18.16±0.14
8.42±0.26
12.36±0.32
14.28±0.24
12.49±0.16
11.64±0.28
16.32±0.34
6
[21]
7
8
9
1
0a
1
0b
1
1
0c
1
1
20
20
10
2a
1
2b
a
All data (test compounds and indomethacin) were significantly different from untreated
control (P < 0.001).
b
Human recombinant COX-2 enzyme
Human COX-1 enzyme from human platelets
Values are means of at least four experiments
c
d
Antimicrobial Screening
All the newly synthesized compounds were evaluated for their antimicrobial activity. The
microdilution susceptibility test in Müller-Hinton Broth (Oxoid) and Sabouraud Liquid Medium
(
Oxoid) were used for the determination of antibacterial and antifungal activity [33]. The Minimal
inhibitory concentrations (MIC g / mL) of test compounds are listed in Table 2.
Table 2. Minimum inhibitory concentrations (MIC g / mL) of test compounds.
6

Compound
MIC (g /mL)
MIC (g / mL)
MIC (g / mL)
Escherichia coli
Staphylococcus aureus Candida albicans
4
6
[21]
5
100
50
>200
100
>200
>200
>200
>200
>200
50
[21]
7
>200
50
>200
100
8
100
50
50
9
>200
>200
100
1
0a
25
>200
100
1
0b
100
100
100
50
1
0c
100
>200
100
1
1
50
1
2a
>200
>200
12.5
---
100
1
2b
>200
25
100
Ampicillin
---
Clotrimazole
---
12.5
Results revealed that compounds 10a exhibited promising antibacterial activity comparable to that
of ampicillin against E. coli, while compounds 5, 7, 9 and 12a displayed half its activity against the
same organism. The test compounds showed weak or no activity against both S. aureus. and C.
albicans.
Acute toxicity
The promising compounds 5, 7 and 10a were further investigated for their oral acute toxicities [34]
and their toxicities through parenteral route [18]. All test compounds were tolerated with
experimental animals up to 80 mg / kg orally. In addition, the same compounds proved to be non-
toxic up to 40 mg / kg when administered through the parentral route.
Structure-Activity Relationship
Concerning some observations for the anti-inflammatory activity, introducing substituents to the
sulfanyl group of 7 appeared to tolerate only methyl group (10a) for a maximum activity. However,
bulkier groups such as ethyl (10b), benzyl (10c), ethylacetate (11), acetonitryl (8), benzoylmethyl
(
12a) or likewise in 12b, 8 and 9 showed an obvious decrease in activity. This observation is
emphasized by the fact that the lowest anti-inflammatory activity is observed for 9 possessing the
7

bulkiest substituent on the sulfanyl group. More insights on some aspects of structure-activity
relationship of the most active compounds are discussed in the Molecular Modeling section.
Interpretation of the antimicrobial results revealed that hydrazino derivative 4 showed weak activity
against E. coli [21]. Conversion of hydrazino derivative 4 into the corresponding ethyl
hydrazinecarboxylate derivative 5 enhanced the antibacterial activity, whereas cyclization of 4 into
the corresponding unsubstituted triazolo derivative 6 did not improve the activity [21]. Cyclization
of compound 4 into the corresponding 1-sulfanyl triazolo derivative 7 enhanced the antibacterial
activity. 1-sulfanyl derivative 7 was found to elicit half the activity of the reference standard against
E. coli. Methylation of 1-sulfanyl derivative 7 to give 10a increased the activity against E. coli to be
comparable to that of the reference standard while its alkylation with phenacyl bromide to give 12a
preserved the activity against E. coli. Generally speaking, bulkier sufanyl substituents than 10a
appeared to enhance the antimicrobial activity while diminishing the anti-inflammatory activity as
appeared in 8, 9, 11 and 12a. However, congeneric 10b and 10c are exceptions from such
observation.
Molecular Modeling
The aim of the molecular modeling investigation in our study is to rationalize the binding scenario
of the compounds with observed biological activity towards COX-2 enzyme.
Based on our previous in silico investigation to select a reliable COX-2 model,[21] we used the
human COX-2 crystal structure (PDB: 5IKQ) for the docking experiments. Interestingly, we
observed a correlation between the docking scores (fitness) of the biologically active compounds (5,
7
and 10a) with their measured activity against human recombinant COX-2, as can be seen in Table
and Table 3. For example, the best docking fitness can be seen for 10a which is also the most
1
active compound against COX-2. In addition, 5 showed superior activity and docking fitness to 7
following 10a. This correlation is clearly seen by the fact that GOLD (ChemPLP) ranked Celecoxib
superior to them all where this observation is also coherent with the biological activity.
Elucidating the observed in vitro selectivity of 5, 7 and 10a towards COX-2 over COX-1 by
docking experiments, we find that these compounds are not able demonstrate a specific binding
scenario in COX-1 binding site. This observation is augmented by the fact that the volume of the
binding site of COX-1 is much smaller than COX-2.[35-37] Besides, the size features (e.g.,
molecular weight) of 5, 7 and 10a are generally higher than the average range of the diverse and
representative COX-1 and COX-2 ligands of DEKOIS 2.0 benchmark sets.[21, 38-40]
The docking poses of the compounds appeared to access more residues in the binding site compared
to the co-crystallized ligand due to core scaffold differences (Figure 1 and Figure 2). The most
active compound 10a showed comparable types of interactions to 7 in the binding site of COX-2
8

explained by their optimum overlay of their docking poses, Figure 2. Such interactions are
inspected to be mainly favorable hydrophobic contacts. For instance, the core tetrahydrobenzo
group is packed between Val 116, Leu 531 and Ser 530, and the central thienopyrimidinone moiety
shows favorable contact with Val 523, apparently as H-arene interaction. Likewise, the sulfanyl
group demonstrates interactions with Leu 352 and Ser 353. The benzyl substituent attached on the
central triazolothienopyrimidinone is packed deeply between Met 522, Phe 518, Leu 384, Trp 387,
Tyr 385, Phe 381 and Leu 352. Additionally, the methylsulfanyl group in 10a is packed in a sub-
pocket formed by Ala 516, Phe 518 and Arg 513 and hence shows extra hydrophobic contacts
compared to the sulfanyl group in 7. This explains the superior docking fitness and activity of 10a.
It is worthy to mention that bulkier substituents on the sulfanyl group than in 10a would
presumably show unfavorable fitting and possibly steric hindrance in the aforementioned sub-
pocket. This rational is consistent with all the tetracyclic system of triazolo[4,3-a]-6,7,8,9-
tetrahydrobenzo[b]thieno[3,2-e]pyrimidinone derivatives which can explain the observed lower
anti-inflammatory activity of bulkier 8, 9, 10b, 10c, 11, 12a and 12b.
One interesting point in the tricyclic system of hydrobenzo[b]thieno[2,3-d]pyrimidinone derivative
5
is that its docking pose demonstrates a vertical flip along the central thienopyrimidinone moiety
compared to 7 and 10a poses, as shown in Figure 3. This flip is necessary to avoid a possible clash
of the extended hydrazine carboxylate group with Arg 513 or His 90 when resembling 7 or 10a
docking pose. As a result, the hydrazino carboxylate of 5 docking pose is directed between Ser 530
and Phe 381 from one side and Tyr 385 and Tyr 348 from another side, and hence the core
thienopyrimidinone moiety is observed to be directed towards Leu 531. Overall, this binding
scenario generated docking fitness better than 7 and less than 10a which is in coherence with the
observed biological activity.
Table 3. Comparison of the docking performance 5, 7 and 10a against COX-2 and COX-1.
COX-2
COX-1
Docking fitness
Celecoxib*
81.95
62.65
68.56
65.70
69.23
53.62
Indomethacin*
59.76
5
7
Non-specific binding
Non-specific binding
Non-specific binding
1
0a
*
Docking results of celecoxib and indomethacin are freely
adapted from our previous study[21]. The docking score is
expressed as fitness of ChemPLP scoring function of GOLD (v
5.2) [41-46].
9

Figure 1. Co-crystal ligand in the binding site of human COX-2 (PDB: 5IKQ) in 3D and 2D
demonstrations for (A) and (B), respectively.
Figure 2. Overlay of the docked poses of compound 7 (green sticks) and 10a (orange sticks) into
the binding site of COX-2 (PDB: 5IKQ) in 3D and 2D demonstrations of 7 for (A) and (B),
respectively.
1
0

Figure 3. Overlay of the docked poses of compound 7 (green sticks) and 5 (red sticks) into the
binding site of COX-2 (PDB: 5IKQ) in 3D and 2D demonstrations of 5 for (A) and (B),
respectively.
Conclusion
The objective of this study is to synthesize thienopyrimidinone and triazolothienopyrimidinone with
dual anti-inflammatory antimicrobial. It could be safely concluded that compounds 5, 7 and 10a
represent promising dual anti-inflammatory anti-microbial agents with minimal ulcerogenic effect
that could act safely up to 80 mg / kg orally or 40 mg / kg parentrally. Their docking experiments
correlated with their biological activity and confirmed the high selectivity binding towards COX-2.
Concerning the antimicrobial screening, compound 10a showed distinguishing inhibitory effect on
the growth of E. coli comparable to that of ampicillin. Also, compounds 5, 7 and 12a possessed
5
0% of the inhibitory activity of ampicillin against E. coli. This work represents a fruitful matrix to
enrich the chemical space for dual anti-inflammatory anti-microbial activities.
Experimental
Chemistry
Melting points were determined in open glass capillaries on a Gallen-kamp melting point apparatus
and are uncorrected. IR spectra were recorded on Perkin-Elmer 1430 infrared spectrophotometer
1
(
Perkin-Elmer, Norwalk, CT, USA). H-NMR spectra were scanned on a Brucker Avance
spectrophotometer (300 MHz) or on Jeol spectrophotometer (500 MHz) using tetramethylsilane
TMS) as internal standard and DMSO-d or the indicated solvents (chemical shifts are given in δ
(
6
ppm). Splitting patterns were designated as follows; s: singlet; d: doublet; t: triplet; m: multiplet.
Mass spectra were run on Finnigan mass spectrometer SSQ/7000 (70 eV) and on Shimadzu GCMS-
QP-1000EX mass spectrometer at 70 ev. Elemental analyses were performed on Vario Elementar,
on Vario EI Fab.-Nr and on Perkin Elmer 2400 CHN Elemental Analyzer and were within ± 0.4%
of the theoretical values. Follow up of the reactions and checking the homogeneity of compounds
were made by ascending TLC run on silica gel 60 (GF 254, Adwic). The ratio of solvent systems
used as eluents were volume to volume. Visualization of spots was performed by exposure to UV
lamp at λ 254 nm for few seconds and by exposure to iodine vapors.
The following compounds were obtained according to previously reported methods: 1 [47-49] and 3
[
24]. Compounds 1, 2, 3, 4 and 6 were previously prepared in our laboratory [21].
1
1

Ethyl N'-(3-benzyl-4-oxo-3,4,5,6,7,8-hexahydrobenzo[b]thieno[2,3-d]pyrimidin-2-yl)hydrazine-
carboxylate 5
Ethyl chloroformate (0.87 g, 0.77 mL, 0.008 mol) was added drop wise to a cold solution of 2-
hydrazino derivative 4 (0.33 g, 0.001 mol) in dry pyridine (10 mL). The reaction mixture was
heated on a water bath for 20 h then cooled and poured onto crushed ice. The obtained precipitate
was separated, washed with water, then with dilute HCl, dried and crystallized from ethanol / water
-
1
(
10:0.5); Mp. 134-136 °C; yield: 85 %; IR (KBr, cm ): 3383, 3262 (NH); 1740 (ester C=O); 1659
C=O); 1620 (C=N); 1589, 1466 (C=C); 1548 (δ NH); 1245, 1068 (υas, υ COC); 1156, 1033 (CSC);
); 1.68-1.87 (m, 4H, 2CH );
); 4.14 (q, J = 6.9 Hz, 2H, CH CH ); 5.29 (s, 2H,
O exchangeable); 6.78 (s, 1H, NH, D O exchangeable); 7.26-7.37
m, 5H, Ar-H); Anal. Calcd. for C20 S (398.48): C, 60.28; H, 5.56; N, 14.06; S, 8.05.
Found: C, 59.89; H, 5.22; N, 13.76; S, 8.04
(
s
1
H-NMR (CDCl
.69, 2.97 (2t, J = 5.35 Hz, each 2H, 2CH
CH ); 6.27 (s, 1H, NH, D
3
, 500 MHz, δ ppm): 1.24 (t, J = 6.9 Hz, 3H, CH
2
CH
3
2
2
2
2
3
2
C
6
H
5
2
2
(
H
22
N
4
O
3
4
5
-Benzyl-1-sulfanyl-1,2,4-triazolo[4,3-a]-6,7,8,9-tetrahydrobenzo[b]thieno[3,2-e]pyrimidin-
(4H)-one 7
Method A:
To a mixture of 2-hydrazino derivative 4 (3.26 g, 0.01 mol) and NaOH (0.4 g, 0.01 mol) in absolute
ethanol (20 mL), carbon disulfide (30 mL) was added. The reaction mixture was heated under
reflux for 18 h and the solvent was then evaporated under reduced pressure. The residue was
dissolved in water, filtered and the filtrate was neutralized with conc. HCl. The formed precipitate
was separated, washed with water, dried and crystallized from dioxane / water (10:0.5); Mp. 282-
2
84 °C; yield: 82 %.
Method B:
An appropriate isothiocyanate derivative, e.g. phenylisothiocyanate or 4-tolylisothiocyanate (0.002
mol), was added to a well stirred suspension of 2-hydrazino derivative 4 (0.65 g, 0.002 mol) in
absolute ethanol (10 mL).The reaction mixture was heated under reflux for 3 h. The obtained solid
was separated while hot, washed with ethanol, dried and crystallized from dioxane / water (10:0.5);
yield: 82 -90 %.
Products obtained employing both methods (A & B) were found to be identical as confirmed by
1
TLC, COTLC, Mp., mixed Mp., IR and H-NMR.
-1
IR (KBr, cm ): 3182 (NH); 2566 (SH); 1661 (C=O); 1618, 1513 (C=N, C=C); 1552, 1334, 1177,
1
9
17 (N-C=S); 1533 (δ NH); 1158, 1069 (CSC); H-NMR (DMSO-d
6
, 300 MHz, δ ppm): 1.64-1.86
(
(
m, 4H, 2CH
2
); 2.75-2.88 (m, 4H, 2CH
2
); 5.14 (s, 2H, CH
2
C
6
H
5
); 7.23-7.42 (m, 5H, Ar-H); 14.05
+
.
s, 1H, SH, D
2
O exchangeable); EI-MS m/z (relative abundance %): 368 (38) (M ); 277 (32);
1
2

9
1
2
1(100); 65 (22); 51 (10); Anal. Calcd. for C18
4.84. Found: C, 57.62; H, 4.21; N, 14.44
H
16
N
4
OS
2
.½ H O (377.49): C, 57.27; H, 4.54; N,
2
-(4-Benzyl-5-oxo-1,2,4-triazolo[4,3-a]-4,5,6,7,8,9-hexahydrobenzo[b]thieno[3,2-e]pyrimidin-1-
ylsulfanyl)acetonitrile 8
A mixture of 1-sulfanyl derivative 7 (0.76 g, 0.002 mol), anhydrous potassium carbonate (0.55 g)
and potassium iodide (0.02 g) in dry acetone (5 mL) was stirred at room temperature for 15
minutes. Chloroacetonitrile (0.23 g, 0.19 mL, 0.003 mol) was then added drop wise with stirring to
the reaction mixture. The mixture was stirred for further 1 h at room temperature and the obtained
precipitate was separated, washed with water, dried and crystallized from dioxane; Mp. 228-230 °C;
-1
yield: 88%; IR (KBr, cm ): 2230 (CN); 1666 (C=O); 1628 (C=N); 1584, 1505 (C=C); 1155, 1079
1
(
CSC); H-NMR (CDCl
3
, 500 MHz, δ ppm): 1.84-1.90 (m, 4H, 2CH
); 3.90 (s, 2H, SCH CN); 5.46 (s, 2H, CH ); 7.27-7.32 (m, 3H, benzyl C 3,4,5-H);
.64 (d, J = 6.85 Hz, 2H, benzyl C2,6-H); EI-MS m/z (relative abundance %): 407 (30) (M ); 368
10); 367 (28); 91 (100); 65 (17); Anal. Calcd. For C20 OS (407.51): C, 58.95; H, 4.20; N,
2
); 2.77-2.79, 3.03-3.05 (2m,
each 2H, 2CH
2
2
2
C
6
H
5
+
.
7
(
H
17
N
5
2
1
7.19. Found: C, 58.55; H, 3.90; N, 16.91.
-Benzyl-1-(5-phenylamino-1,3,4-thiadiazol-2-ylmethylsulfanyl)-1,2,4-triazolo[4,3-a]-6,7,8,9-
4
tetrahydrobenzo[b]thieno[3,2-e]pyrimidin-5(4H)-one
A mixture of 2-sulfanylacetonitrile derivative 8 (0.43 g, 0.001 mol) and phenylthiosemicarbazide
0.17 g, 0.001 mol) in glacial acetic acid (5mL) containing few drops of conc. H SO was heated
9
(
2
4
under reflux for 12 h and left to cool. The reaction mixture was poured onto crushed ice and the
obtained precipitate was separated, washed with water, dried and crystallized from chloroform /
-1
ethanol (1:1), Mp. 206-208 °C; yield: 89%; IR (KBr, cm ): 3240 (NH); 1674 (C=O); 1587,
1
1
507(C=N, C=C); 1551 (δ NH); 1157,1080 (CSC); H-NMR (CDCl
3
, 500 MHz, δ ppm): 1.79-1.92
); 3.98 (s, 2H, SCH ); 5.42 (s, 2H,
); 7.26-7.34 (m, 9H, benzyl C3,4,5-H, phenyl-H, NH, D O exchangeable), 7.63 (d, J = 7.65
(557.72): C, 58.15; H, 4.16; N, 17.58.
(
m, 4H, 2CH
2
); 2.72-2.85, 2.98-3.10 (2m, each 2H, 2CH
2
2
CH
2
C
6
H
5
2
Hz, 2H, benzyl C2,6-H); Anal. Calcd. for C27
Found: C, 58.35; H, 4.28; N, 17.40.
H
23
N
7
OS
3
4
-Benzyl-1-substituted
sulfanyl-1,2,4-triazolo[4,3-a]-6,7,8,9-tetrahydrobenzo[b]thieno[3,2-e]-
pyrimidin-5(4H)-ones 10a-c
A solution of 1-sulfanyl derivative 7 (0.38 g, 0.001 mol) in NaOH 10% (10 mL) was treated with
the proper alkyl iodide (0.0015 mol) or benzyl chloride (0.001 mol). The reaction mixture was
stirred for 4 h at room temperature. The obtained precipitate was separated, washed with water,
dried and crystallized from chloroform / ethanol (2:1).
-1
IR (KBr, cm ) of compounds 10a-c: 1659-1653 (C=O); 1629-1626 (C=N); 1580-1573, 1500-1493
C=C); 1157-1148, 1072-1065 (CSC).
(
1
3

4
-Benzyl-1-methylsulfanyl-1,2,4-triazolo[4,3-a]-6,7,8,9-tetrahydrobenzo[b]thieno[3,2-e]-
pyrimidin-5(4H)-one 10a
Mp. 270-272 °C; yield: 88%; H-NMR (CDCl
s, 3H, SCH ); 2.76, 3.03 (2t, J = 6.1 Hz, each 2H, 2CH
H, benzyl C3,4,5-H), 7.65 (d, J =7.65 Hz, 2H, benzyl C2,6-H); Anal. Calcd. for C19
382.50): C, 59.66; H, 4.74; N, 14.65. Found: C, 60.06; H, 4.40; N, 14.59.
1
3
, 500 MHz, δ ppm): 1.83-1.89 (m, 4H, 2CH
); 5.43 (s, 2H, CH ); 7.26-7.30 (m,
OS
2
); 2.69
(
3
2
2
C
6
H
5
3
H
18
N
4
2
(
4
-Benzyl-1-ethylsulfanyl-1,2,4-triazolo[4,3-a]-6,7,8,9-tetrahydrobenzo[b]thieno[3,2-e]pyrimidin-
5
(4H)-one 10b
1
Mp. 216-218 °C; yield: 80%; H-NMR (DMSO-d
6
, 500 MHz, δ ppm): 1.22 (t, J = 7.65 Hz, 3H,
); 2.99 (q, J = 7.65
); 7.24-7.29 (m, 5H, benzyl H);EI-MS m/z (relative
abundance %):396 (21) (M ); 395 (14); 368 (11); 277 (11); 92 (11); 91(100); 90 (11); 65 (30);
Anal. Calcd. for C20 OS (396.53): C, 60.58; H, 5.08; N, 14.13. Found: C, 60.27; H, 4.76; N,
CH
2
CH
3
); 1.72-1.75 (m, 4H, 2CH
2
); 2.69, 2.84 (2t, J = 6.1 Hz, each 2H, 2CH
2
Hz, 2H, CH
2
CH ); 5.23 (s, 2H, CH
3
2
C
6
H
5
+
.
H
20
N
4
2
1
4
5
4.05.
-Benzyl-1-benzylsulfanyl-1,2,4-triazolo[4,3-a]-6,7,8,9-tetrahydrobenzo[b]thieno[3,2-e]pyrimidin-
(4H)-one 10c
1
Mp. 192-194 °C; yield: 78 %; H-NMR (DMSO-d
6
, 500 MHz, δ ppm): 1.70-1.76 (m, 4H, 2CH
); 4.26 (s, 2H, SCH ); 5.27 (s, 2H, CH ); 7.17-
.36 (m, 10H, 2 x benzyl H); Anal. Calcd. for C25 OS (458.60): C, 65.47; H, 4.84; N, 12.22.
2
);
2
7
.69, 2.83 (2t, J = 5.35 Hz, each 2H, 2CH
2
2
C
6
H
5
2
C
6
H
5
H
22
N
4
2
Found: C, 65.80; H, 4.65; N, 12.16
Ethyl 2-(4-Benzyl-5-oxo-1,2,4-triazolo[4,3-a]-4,5,6,7,8,9-hexahydrobenzo[b]thieno[3,2-e]-
pyrimidin-1-ylsulfanyl)acetate 11
Method A:
A mixture of 1-sulfanyl derivative 7 (0.38 g, 0.001 mol), anhydrous potassium carbonate (0.21 g)
and ethyl chloroacetate (0.12 g, 0.11 mL, 0.001 mol) in dry dimethylformamide (10 mL) was heated
under reflux for 3 h. The reaction mixture was allowed to cool and poured onto ice-cold water. The
obtained precipitate was separated, washed with water, dried and crystallized from dioxane; Mp.
2
32-234 °C; yield: 84 %.
Method B:
Sodium ethoxide solution was prepared by dissolving sodium metal (0.03 g, 0.001 g atom) in
absolute ethanol (10 mL). 1-sulfanyl derivative 7 (0.38 g, 0.001 mol) was dissolved in this solution
then ethyl bromoacetate (0.17 g, 0.11 mL, 0.001 mol) was added drop wise with stirring. The
reaction mixture was heated under reflux for 3 h and allowed to cool. The obtained precipitate was
separated, washed with ethanol then with water, dried and crystallized from dioxane; Mp. 232-234
1
4

°C; yield: 82 %. The product obtained from method A was found to be identical to that obtained
from method B as revealed by TLC, COTLC, Mp., mixed Mp. and IR.
-1
IR (KBr, cm ): 1743 (ester C=O); 1673 (C=O); 1608, 1590, 1514 (C=N, C=C); 1241, 1080 (υas, υ
s
1
COC); 1120, 1025 (CSC); H-NMR (DMSO-d
6
, 500 MHz, δ ppm): 1.17 (t, J = 6.85 Hz, 3H,
); 2.74, 2.88 (2t, J = 6.1 Hz, each 2H, 2CH ); 4.13 (q, J = 6.85
); 4.27 (s, 2H,SCH ); 5.24 (s, 2H, CH ); 7.18-7.37 (m, 5H, benzyl H); EI-MS
m/z (relative abundance %): 454 (31) (M ); 125 (13); 104 (18); 93 (12); 92 (10); 91 (100); 90 (11);
8 (24); 77 (20); 76 (12); 70 (11); 65 (38); 63 (14); 51 (37); 50 (15); Anal. Calcd. for C22
454.57): C, 58.13; H, 4.88; N, 12.33. Found: C, 58.43; H, 4.58; N, 12.11.
CH
2
CH
3
); 1.73-1.77 (m, 4H, 2CH
CH
2
2
Hz, 2H, CH
2
3
2
2
C
6
H
5
+
.
7
H
22
N
4
O S
3 2
(
4
-Benzyl-1-(4-substituted benzoylmethylsulfanyl)-1,2,4-triazolo[4,3-a]-6,7,8,9-tetrahydrobenzo-
[
b]thieno[3,2-e]pyrimidin-5(4H)-ones 12a and 12b
A mixture of 1-sulfanyl derivative 7 (0.38 g, 0.001mol) and the appropriate phenacyl bromide
0.001 mol) in absolute ethanol (10 mL) was heated under reflux for 5 h. The reaction mixture was
(
then allowed to cool to room temperature. The solid separated was filtered, washed with ethanol,
dried and crystallized from chloroform / ethanol (2:1).
-1
IR (KBr, cm ) of compounds 12a and 12b: 1676 -1675 (benzoyl C=O); 1664-1660 (pyrimidinone
C=O); 1587-1582, 1483-1482 (C=N, C=C); 1204-1201, 1076-1071 (CSC); 546 (C-Br) for
compound 12b.
4
-Benzyl-1-(4-benzoylmethylsulfanyl)-1,2,4-triazolo[4,3-a]-6,7,8,9-tetrahydrobenzo[b]thieno[3,2-
e]pyrimidin-5(4H)-one 12a
Mp. 210-212 °C; yield: 94%; H-NMR (DMSO-d
.66, 2.84 (2t, J = 5.35 Hz, each 2H, 2CH ); 4.82 (s, 2H, SCH
m, 8H, 5 benzyl H & 3 benzoyl C 3,4,5-H); 7.86 (d, J = 7.65 Hz, 2H, benzoyl C2,6-H); Anal. Calcd.
for C26 (486.61): C, 64.17; H, 4.56; N, 11.51. Found: C, 64.09; H, 4.95; N, 11.12
-Benzyl-1-(4-(p-bromobenzoyl)methylsulfanyl)-1,2,4-triazolo[4,3-a]-6,7,8,9-tetrahydrobenzo[b]-
1
6
, 500 MHz, δ ppm): 1.71-1.74 (m, 4H, 2CH
2
);
2
2
2
); 5.26 (s, 2H, CH ); 7.21-7.60
2
C
6
H
5
(
H
22
N
4
O S
2 2
4
thieno[3,2-e]pyrimidin-5(4H)-one 12b
1
Mp. 226-228 °C; yield: 91%; H-NMR (DMSO-d
6
, 300 MHz, δ ppm): 1.75-1.82 (m, 4H, 2CH
); 5.31 (s, 2H, CH ); 7.24-7.35
m, 3H, benzyl C3,4,5-H), 7.41 (d, J = 7.2 Hz, 2H, benzyl C2,6-H); 7.68 (d, J = 8.1 Hz, 2H, benzoyl
3,5-H); 7.82 (d, J = 8.1 Hz, 2H, benzoyl C2,6-H); EI-MS m/z (relative abundance %): 381(15); 368
16); 277 (12); 222 (10); 185 (16); 183 (16); 91 (100); 65 (14); Anal. Calcd. for C26
565.51): C, 55.22; H, 3.74; N, 9.91. Found: C, 55.18; H, 3.43; N, 9.92.
2
);
2
.71- 2.78, 2.90-2.94 (2m, each 2H, 2CH
2
); 4.80 (s, 2H, SCH
2
2
C
6
H
5
(
C
(
(
H
21BrN
4
O
S
2 2
Biological screening
Anti-inflammatory screening
1
5

Cotton pellet-induced granuloma bioassay[29]
Adult male Sprague-Dawley rats (Faculty of Pharmacy animal house, Alexandria University,
Egypt) (120-140 g) were used to evaluate the anti-inflammatory efficacy of the synthesized
compounds. All procedures were performed as described earlier.[50] Results are shown in Table 1.
Significant difference between the control and treated groups was performed using Student’s t-test
and P values were considered significant at P < 0.001.
Ulcerogenic activity[30]
Male albino rats (Faculty of Pharmacy animal house, Alexandria University, Egypt) (100 -120 g)
were fasted for 12 h prior to the administration of compounds. Water was given ad libitum. Rats
were distributed into groups, six animals each. All steps were conducted as reported earlier [21].
The ulcer index which indicates the severity of the stomach lesions was calculated using an
arbitrary scale. The percentage ulceration (Table 1) for each group was calculated as follows:
%
Ulceration = (Number of animals bearing ulcer in a group / Total Number of animals in the
same group) x 100
Human COX-1 and COX-2 enzymatic assay[34]
Human COX-1 and COX-2 activities were determined by applying the methodology described by
Wakitani et al.[31] COX-1 assay was carried out using human platelets microsomal fraction
prepared from NSAID-free normal human volunteers as described by Hammarström and
Falardeau.[32] The human recombinant COX-2 was purchased and the experimental procedures to
determine the enzymatic activities were performed as reported earlier.[50] The concentration of the
compound causing 50 % inhibition (IC50) was calculated (Table 1).
Antimicrobial Screening
The microdilution susceptibility test in Müller-Hinton Broth (Oxoid) and Sabouraud Liquid
Medium (Oxoid) were used for the determination of antibacterial and antifungal activity [33]. All
experimental steps were conducted as reported earlier [21]. The results are shown in Table 2.
Acute toxicity
The promising compounds 5, 7 and 10a were further investigated for their oral acute toxicities in
male mice (each 20 g, Faculty of Pharmacy animal house, Alexandria University, Egypt) as
reported [34]. Eight groups of rats each consisting of six animals were used. The compounds were
1
6

given orally suspended in 1% gum acacia, in doses 1, 10, 40, 50, 60, 70, 80 and 100 mg / Kg. 24
hours later, % mortality in each group for each compound was recorded.
Moreover, the selected compounds were tested for their toxicities through parenteral route [18].
Groups of six rats each were used. The compounds or their vehicle propylene glycol (control) were
given as intraperitoneal injection in doses 10, 20, 25, 30, 35, 40 and 50 mg / Kg, respectively.
Survival was followed up to 7 days. The results are summarized in Table 1.
Molecular Modeling
Preparation of target proteins
The coordinates of the X-ray crystal structure of of COX-1 (PDB ID: 1EQG) and COX-2 (PDB ID:
5
IKQ) were obtained from Protein Data Bank and used directly from our previous study. [21]
Preparation of the selected compounds for docking
The compounds were built and prepared by MOE. Generation of meaningful protonation states,
energy minimization steps and calculation of partial charges were conducted as reported earlier
[
21]. The prepared molecules were then saved as SD file for the docking runs.
Docking experiments
GOLD (version 5.2) [41-44] was used for employing the scoring functions ChemPLP for the
docking experiments against COX-1 and COX-2 models. The definition of the binding site and
search efficiency were conducted as reported earlier.[21] All graphical representations in Figure 1-3
were rendered by Molecular Operating Environment (MOE). [51]
Acknowledgement
The authors wish to thank Dr. Elsayed aboulmagd, department of Microbiology, Faculty of
Pharmacy, University of Alexandria, Egypt for his assistance during antimicrobial testing. TMI
acknowledges Prof. Dr. Frank Boeckler, Lab for Molecular Design & Pharmaceutical Biophysics,
Eberhard Karls University Tuebingen, Germany, for granting access to the computational tools.
Conflict of interest
No conflict of interest.
Ethical conduct of research
The protocols used in the present study followed the guidelines set in ‘The Guide for the Care and
Use of Laboratory Animals’, and got approval By ACUC, Faculty of Pharmacy, Alexandria
University, Project No. 22 at 22/5/2013. Copy of approval is available upon request.
1
7

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Graphical abstract
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1

Research highlights:




Novel thienopyrimidinone and triazolothienopyrimidinone derivatives were synthesized.
In vitro Anti-inflammatory, and antimicrobial activity screening were performed.
In vivo acute toxicity test was performed to ensure the safety of oral and parentral routes.
The in silico experiments rationalized the anti-inflammatory activity observed and correlated
with the observed biological activity.

Some compounds showed promising dual anti-inflammatory antimicrobial activity.
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